This grant has been continuously funded since 1996, the most recent cycle started in 09/2007. The focus will remain on how specific cell cycle proteins regulate glomerular cells in health, and in disease. Glomerular diseases are an important field of study as they are the commonest causes of chronic and end stage kidney disease. In this competitive renewal, we will continue to study the glomerular podocytes (podo), because of their critical biological role in the prevention of proteinuria and sclerosis. Major progress made during the current funding cycle was our discovery of a new paradigm relating to cell cycle control, where the constitutively expressed cyclin dependent kinase (cdk) 5 is dually activated by the relatively unknown cyclin I, as well as the non-cyclin p35. The second major discovery made was that activation of cdk5 by cyclin I and p35 safeguards terminally differentiated cells like podo and neurons from apoptosis. The clinical significance is podo loss is a major contributor to proteinuria and sclerosis. The impact and relevance of cdk5, cyclin I and p35 in the kidney was further supported by our preliminary data showing that mice deficient in cdk5, or lacking both cyclin I and p35, do not develop normal glomeruli. Although cell cycle proteins are traditionally viewed as regulating cell proliferation, and more recently apoptosis, the goal of the first aim of this competitive renewal is to show a new paradigm for cell cycle protein function, based on our preliminary data. The proposed studies will test the hypothesis that cdk5 and its activators cyclin I and p35 control proteinuria and podo shape by complexing with, phosphorylating and regulating levels of the slit diaphragm proteins podocin and nephrin, and the actin-binding proteins podocalyxin and ezrin. These studies will change our current thinking by closing the gap on our current knowledge of how key podo proteins are governed, and what happens in glomerular diseases. The overall goal of the second aim is to show new regulatory paradigms for cyclin I-p35-cdk5, which play a fundamental role in glomerular and brain development. Following up on our preliminary data, this aim will test two hypotheses: first, that cyclin I anchors cdk5 to the nucleus, whereas p35 insures a cytoplasmic localization;second, that the dual activation by cyclin I and p35 insures constant cdk5 activity, and both cooperate to set the threshold for cdk5 functions under normal and disease states. We have developed innovative tools to test these hypotheses by generating transgenic mice lacking cdk5 specifically in podo (Nephrin-cre-EGFP-cdk5 flox), lacking cdk5 specifically in podo in an inducible manner (NPHS2rtTA/TetO-cre mice), and lacking both activators (cyclin I-/-p35-/- mice). Cultured podo from these mice will complement the approach. In summary, the studies proposed will advance scientific knowledge in glomerular diseases, and in cell cycle protein research. Our published work shows that studying podo has the added advantage that discoveries in cell cycle can be broadly applied to other terminally differentiated cells, which helps drive discovery in the field. PUBLIC HEALTH RELEVANCE: The studies proposed in this competitive renewal will show novel roles in podo for cdk5 and its partners cyclin I and p35, in the regulation of the slit diaphragm proteins podocin and nephrin, and the actin-regulating proteins podocalyxin and ezrin, and how these are affected in disease.